Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Editorial Note: This manuscript has been previously reviewed at another journal that is not operating a transparent peer review scheme. This document only contains reviewer comments and rebuttal letters for versions considered at Nature Communications.

REVIEWERS' COMMENTS
Reviewer #1 (Remarks to the Author): The authors have provided information on viral genome sequence assembled in the study, and revised the manuscript in response to my concerns. The following points should be further clarified: 1. Are the meta-transcriptomic sequencing and viral genome assembly of the samples from bats, rodents, insectivores, pikas, pangolins, and zoo animals conducted respectively or in the same batch? Are any controls included in the sequencing? 2. As previously mentioned, the SARS-CoV-2-related coronavirus sequences in Figure 2A have been published by Xiao et al (Nature 2020;583:286-289). I don't think the published data is allowed to include in analysis as a new finding in this manuscript. 3. Many previously reported viral sequences in the same animal species have been used in phylogenetic trees in this manuscript. These important publications should be cited, and authors do not need to avoid these similar studies.
Reviewer #3 (Remarks to the Author): The revisions are suitable for this reviewer.
1. Are the meta-transcriptomic sequencing and viral genome assembly of the samples from bats, rodents, insectivores, pikas, pangolins, and zoo animals conducted respectively or in the same batch? Are any controls included in the sequencing?
Reply: As the animals used in this study were collected over a long period of time, the sequencing and genome assembly of these samples were processed in many batches. We added information of batches in Table S1 (Supplementary Data 1 in this updated manuscript).
When sequencing multiple samples in the same batch, each sample's library was assigned to a separate channel to prevent contamination and cross-talk between the libraries. Robotic arms are used throughout the sequencing process to prevent errors and contamination caused by human error. As we responded in the previous revision, Reply: As we responded in the previous revision, we did not use the published data as a new finding in this manuscript. Although some of the pangolins sequenced in this study are from the same batch of pangolins used in our previous study (Xiao et al., Nature 2020;583:286-289), 55 meta-transcriptomes were sequenced in this study to attain their viromes rather than in Xiao et al. In addition to the pangolin CoV, we found many other viruses in pangolins (Figs 3 and 4). In all of the topologies (Figs   2-6), viruses assembled in this study were marked in colors to distinguish from published data. Thus the three pangolin CoVs assembled in this study were marked in red in Fig 2A,  3. Many previously reported viral sequences in the same animal species have been used in phylogenetic trees in this manuscript. These important publications should be cited, and authors do not need to avoid these similar studies.

Reply:
We respect the contributions of these previous studies and have cited 71 references in the main text. As we responded in the previous revision, we list the references for the reported viral sequences that were used in our phylogenetic trees in Table S8. There are 332 references. We are very sorry that it is impossible to list this many references in the main text, thus only some of them are cited in the main text, while the others are only listed in Table S8 (Supplementary Data 6 in this updated manuscript).